Mass spectrometry (MS) is a sensitive method for the simultaneous identification and relative quantitation of thousands of proteins between multiple samples. On-going advancements in MS instrumentation continue to push the ability of the scientific community to characterize the protein dynamics in complex biological systems to a great depth. Despite MS capabilities, a majority of MS samples are analyzed without significant pre-injection characterization or normalization, because current methods to monitor and measure proteins, such as UV absorption or bichinconic acid (BCA) assays, work poorly with peptides, consume too much valuable sample, and lack the required sensitivity. This lack of sample characterization and normalization leads to difficulties with standardization and reproducibility of MS experiments and significant under-productive instrument time.
Commercially available colorimetric protein and peptide solution quantitation methods include biuret (Gornall et al. J. Biol. Chem. 177 (1949) 751), Lowry (Lowry et al. J. Biol. Chem. 193 (1951) 265), bicinchoninic acid (BCA) (Smith et al. Anal. Biochem. 150 (1985) 76), Coomassie Blue G-250 dye-binding (Bradford, Anal. Biochem. 72 (1976) 248), and colloidal gold (Stoscheck, Anal. Biochem. 160 (1987) 301).
The biuret method is based on a protein forming a complex with cupric ions. Peptide nitrogen binds to copper (II) ion under alkaline conditions, producing a purple color. The absorption maximum of the product is 550 nm. The sensitivity is 1 mg protein/ml to 6 mg protein/ml. The biuret method is a relatively insensitive protein determination method compared to other commercial methods of colorimetric protein determination.
Another method combines the biuret reaction and the copper(1)-bathocuproine chelate reaction (Determination of Proteins by a Reverse Biuret Method Combined with the Copper-Bathocuproine Chelate Reaction. Clinica Chimica Acta., 216 (1993) 103-111). In this method, a sample protein forms a Cu2+-protein chelate complex (biuret reaction) during the first step. Excess Cu2+ is reduced to Cu+ by ascorbic acid, allowing Cu+ to form a Cu+-bathocuproine chelate complex during the second step. The amount of Cu+-bathocuproine chelate complex formed is inversely proportional to the protein concentration. This is a negative or indirect assay using a bathocuproine chelator to determine protein concentration.
The Lowry method is a modified biuret reaction. It occurs in two steps: first, peptide bonds react with copper(II) ions under alkaline conditions, then Folin-Ciocalteau phosphomolybdic-phosphotungstic acid reduces to heteropolymolybdenum blue by copper-catalyzed oxidation of aromatic amino acids. The absorption maximum of the product is 750 nm. The Lowry method is more sensitive than the biuret method, with a linear sensitivity of 0.1 mg protein/ml to 1.5 mg protein/ml for bovine serum albumin (BSA). Certain amino acids, detergents, lipids, sugars, and nucleic acids interfere with the reaction. The reaction is pH dependent and pH should be maintained between pH 10 and pH 10.5.
The BCA method is related to the Lowry method in that peptide bonds in proteins first reduce cupric ion (Cu2+) to produce a tetradentate-cuprous ion (Cu1+) complex in an alkaline medium. The cuprous ion complex then reacts with BCA (2 molecules BCA per Cu1+) to form an intense purple color that can be measured at 562 nm. The BCA-Copper reaction is shown below:
Because BCA is stable in alkaline medium, the BCA method can be carried out in one step, compared to two steps needed in the Lowry method. The BCA method better tolerates potential inhibitory or interfering compounds in the sample compared to the Lowry method. For example, up to 5% of each of sodium dodecyl sulfate (SDS), Triton X-100, and Tween-20 can be present and not interfere with the BCA method, compared to only 1% SDS, 0.03% Triton X-100, and 0.062% Tween-20 that can be present and not interfere with the Lowry method. The BCA method also has increased sensitivity and an expanded linear working range compared to the Lowry method.
A MICRO BOA™ Protein Assay Kit (Thermo Fisher Scientific) permits quantitation of dilute sample solutions (0.5 μg/ml to 20 μg/ml) by using larger sample volumes to obtain higher sensitivity. Despite the increased sensitivity, sample volume requirements limit or prevent its use for quantitation of many peptide samples.
A modified BCA assay to quantitate peptides (Kapoor et al. Analytical Biochemistry, 393 (2009) 138-140) acknowledges difficulties measuring peptide concentrations because of high interpeptide variation largely because of peptide hydrophobicity. The modified BCA method estimates peptide concentration by denaturing peptides by treatment at 95° C. for five minutes in the presence of SDS prior to incubation with the BCA working reagent. However, data below 500 μg/ml is very close to noise level and thus is not reliable.
U.S. Pat. No. 4,839,295 discloses using bicinchoninic acid as a chelator to detect proteins, measuring absorbance at 562 nm.
The colloidal gold method is the most sensitive among the colorimetric protein determination methods. Its sensitivity is about 2 μg/ml to 20 μg/ml protein. However, there is significant protein-to-protein variation. Protein binding to colloidal gold causes a shift in colloidal gold absorbance that is proportional to the amount of protein in solution. Most common reagents other than thiols and sodium dodecyl sulfate (SDS) are compatible with the colloidal gold method.
The Coomassie Blue G-250 dye-binding method is based on the immediate absorbance shift from 470 nm to 595 nm that occurs when Coomassie Blue G-250 binds protein in an acidic medium. Color development is rapid and the assay can be performed in ten minutes. The Coomassie Blue G-250 dye-binding method is comparatively free from interference by common reagents except detergents. There is moderate protein-to-protein variation and the method does not work well with peptides.
A total protein assay (Sozgen et al., Talanta, 68 (2006) 1601-1609 Spectrophotometric total protein assay with copper (II) neocuproine reagent in alkaline medium) uses copper(II)-neocuproine (Nc) reagent in alkaline medium with a hydroxide-carbonate-tartarate solution, with neocuproine as chelator. After 30 min incubation at 40° C., absorbance of the reduction product, Cu(I)-Nc complex, is read at 450 nm against a reagent blank. This assay has limited sensitivity because of the limited solubility of neocuproine in alkaline aqueous solution.
U.S. Pat. No. 5,693,291 discloses a quantitative protein method. The method is an indirect two-step method. It uses two reagents: reagent A (tartrate solution and copper sulfate) and reagent B (reducing agent, e.g., ascorbic acid, and bathocuproine chelator). Reagent A contains 0.7 to 2 mmol/I Cu2+ ions and 2 to 4 mmol/I tartrate in alkaline solution. Reagent B contains 1 to 1.5 mmol/I ascorbic acid and 0.5 to 0.8 mmol/I bathocuproine. The proportion of reagent A to reagent B is 1:8 to 1:12, i.e., 1 part reagent A to 8-12 parts reagent B. The combined volume of reagent A and reagent B is between 750 μl and 3000 μl, which is relatively large. Step one of the method mixes 100 μl Reagent A to 50 μl sample, followed by incubating at room temperature for 5 min to 60 min. Step two of the method adds 1 ml reagent B to the step one mixture, followed by brief mixing and reading at 485 nm. This negative or indirect assay quantitates protein by the difference in absorbance in the pre-versus post-bathocuproine chelated sample. It is thus less accurate than a positive or direct assay that quantitates protein directly. It also uses a large volume of standard protein to reagent (volume standard protein to reagent A is 1:1.6 to 1:2.4).
The method provided herein overcomes such drawbacks and provides additional benefits.